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miR-93 promotes cell proliferation in gliomas through activation of PI3K/Akt signaling pathway.

Jiang L, Wang C, Lei F, Zhang L, Zhang X, Liu A, Wu G, Zhu J, Song L - Oncotarget (2015)

Bottom Line: Multiple regulators, such as phosphatase and tensin homolog (PTEN) and PH domain leucine rich repeat protein phosphatases (PHLPP), have also found to be involved in suppression of the PI3K/Akt signaling pathway.Furthermore, we found that overexpressing miR-93 promoted, but inhibition of miR-93 reduced, glioma cell proliferation and cell-cycle progression.Therefore, our results suggest that miR-93 might play an important role in glioma progression and uncover a novel mechanism for constitutive PI3K/Akt activation in gliomas.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathophysiology, Guangzhou Medical University, Guangzhou 510182, China.

ABSTRACT
The PI3K/Akt signaling pathway is frequently activated in various human cancer types and plays essential roles in development and progression of cancers. Multiple regulators, such as phosphatase and tensin homolog (PTEN) and PH domain leucine rich repeat protein phosphatases (PHLPP), have also found to be involved in suppression of the PI3K/Akt signaling pathway. However, how suppressive effects mediated by these regulators are concomitantly disrupted in cancers, which display constitutively activated PI3K/Akt signaling, remains puzzling. In the present study, we reported that the expression of miR-93 was markedly upregulated in glioma cell lines and clinical glioma tissues. Statistical analysis revealed that miR-93 levels significantly correlated with clinicopathologic grade and overall survival in gliomas. Furthermore, we found that overexpressing miR-93 promoted, but inhibition of miR-93 reduced, glioma cell proliferation and cell-cycle progression. We demonstrated that miR-93 activated PI3K/Akt signaling through directly suppressing PTEN, PHLPP2 and FOXO3 expression via targeting their 3'UTRs. Therefore, our results suggest that miR-93 might play an important role in glioma progression and uncover a novel mechanism for constitutive PI3K/Akt activation in gliomas.

No MeSH data available.


Related in: MedlinePlus

PTEN, PHLPP2, and FOXO3 are direct targets of miR-93 in glioma cells(A) Schematic putative target sites of miR-93 in 3′UTRs of PTEN, PHLPP2 and FOXO3, and the sequence of miR-93 mutant (performed as miR-93-mut). (B) Western blotting analysis of the protein levels of PTEN, PHLPP2 and FOXO3 in the indicated cells. (C) Luciferase assay of pGL3-PTEN, PHLPP2 or FOXO3–3′UTR reporter co-transfected with miR-93, miR-93 inhibitor or the control in the indicated cells. Experiments were repeated at least 3 times with similar results, and error bars represent ± SD, *P < 0.05.
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Figure 4: PTEN, PHLPP2, and FOXO3 are direct targets of miR-93 in glioma cells(A) Schematic putative target sites of miR-93 in 3′UTRs of PTEN, PHLPP2 and FOXO3, and the sequence of miR-93 mutant (performed as miR-93-mut). (B) Western blotting analysis of the protein levels of PTEN, PHLPP2 and FOXO3 in the indicated cells. (C) Luciferase assay of pGL3-PTEN, PHLPP2 or FOXO3–3′UTR reporter co-transfected with miR-93, miR-93 inhibitor or the control in the indicated cells. Experiments were repeated at least 3 times with similar results, and error bars represent ± SD, *P < 0.05.

Mentions: In an attempt to identify the mRNA targets of miR-93, we performed a bioinformatics analysis using the publicly available algorithm (TargetScan 6.2). As shown in Figure 4A, PTEN and PHLPP2, which are the inhibitors of PI3K/Akt signaling pathway, and FOXO3, which critical regulator of cell-cycle, were found to be potential targets of miR-93. Western blotting analysis showed that ectopic expression of miR-93 dramatically decreased, whereas inhibition of miR-93 increased, the protein expression of PTEN, PHLPP2 and FOXO3 in both LN18 and Hs683 glioma cells (Figure 4B). Furthermore, when co-transfected with PTEN-, PHLPP2-, or FOXO3-3′UTR dual luciferase reporter plasmid, together with miR-93, miR-93 inhibitor or negative control, into the glioma cells, as shown in Figure 3C, miR-93 led to a consistent reduction of luciferase activity of PTEN, PHLPP2, FOXO3 reporters. Whereas transfection with miR-93 inhibitor upregulated the luciferase activity, respectively (Figure 4C). Of note, the luciferase activity was insusceptible by miR-93-mut (miR-93 mutant) transfection instead of miR-93 (Figure 4C). Besides, the PTEN, PHLPP2 or FOXO3 3′-UTR-luciferase reporter with a mutant miR-93 binding site seed sequence was not inhibited by ectopic expression of miR-93 (Supplementary Figure 2A and 2B). Taken together, the results confirm that PTEN, PHLPP2 and FOXO3 are direct targets of miR-93.


miR-93 promotes cell proliferation in gliomas through activation of PI3K/Akt signaling pathway.

Jiang L, Wang C, Lei F, Zhang L, Zhang X, Liu A, Wu G, Zhu J, Song L - Oncotarget (2015)

PTEN, PHLPP2, and FOXO3 are direct targets of miR-93 in glioma cells(A) Schematic putative target sites of miR-93 in 3′UTRs of PTEN, PHLPP2 and FOXO3, and the sequence of miR-93 mutant (performed as miR-93-mut). (B) Western blotting analysis of the protein levels of PTEN, PHLPP2 and FOXO3 in the indicated cells. (C) Luciferase assay of pGL3-PTEN, PHLPP2 or FOXO3–3′UTR reporter co-transfected with miR-93, miR-93 inhibitor or the control in the indicated cells. Experiments were repeated at least 3 times with similar results, and error bars represent ± SD, *P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4480752&req=5

Figure 4: PTEN, PHLPP2, and FOXO3 are direct targets of miR-93 in glioma cells(A) Schematic putative target sites of miR-93 in 3′UTRs of PTEN, PHLPP2 and FOXO3, and the sequence of miR-93 mutant (performed as miR-93-mut). (B) Western blotting analysis of the protein levels of PTEN, PHLPP2 and FOXO3 in the indicated cells. (C) Luciferase assay of pGL3-PTEN, PHLPP2 or FOXO3–3′UTR reporter co-transfected with miR-93, miR-93 inhibitor or the control in the indicated cells. Experiments were repeated at least 3 times with similar results, and error bars represent ± SD, *P < 0.05.
Mentions: In an attempt to identify the mRNA targets of miR-93, we performed a bioinformatics analysis using the publicly available algorithm (TargetScan 6.2). As shown in Figure 4A, PTEN and PHLPP2, which are the inhibitors of PI3K/Akt signaling pathway, and FOXO3, which critical regulator of cell-cycle, were found to be potential targets of miR-93. Western blotting analysis showed that ectopic expression of miR-93 dramatically decreased, whereas inhibition of miR-93 increased, the protein expression of PTEN, PHLPP2 and FOXO3 in both LN18 and Hs683 glioma cells (Figure 4B). Furthermore, when co-transfected with PTEN-, PHLPP2-, or FOXO3-3′UTR dual luciferase reporter plasmid, together with miR-93, miR-93 inhibitor or negative control, into the glioma cells, as shown in Figure 3C, miR-93 led to a consistent reduction of luciferase activity of PTEN, PHLPP2, FOXO3 reporters. Whereas transfection with miR-93 inhibitor upregulated the luciferase activity, respectively (Figure 4C). Of note, the luciferase activity was insusceptible by miR-93-mut (miR-93 mutant) transfection instead of miR-93 (Figure 4C). Besides, the PTEN, PHLPP2 or FOXO3 3′-UTR-luciferase reporter with a mutant miR-93 binding site seed sequence was not inhibited by ectopic expression of miR-93 (Supplementary Figure 2A and 2B). Taken together, the results confirm that PTEN, PHLPP2 and FOXO3 are direct targets of miR-93.

Bottom Line: Multiple regulators, such as phosphatase and tensin homolog (PTEN) and PH domain leucine rich repeat protein phosphatases (PHLPP), have also found to be involved in suppression of the PI3K/Akt signaling pathway.Furthermore, we found that overexpressing miR-93 promoted, but inhibition of miR-93 reduced, glioma cell proliferation and cell-cycle progression.Therefore, our results suggest that miR-93 might play an important role in glioma progression and uncover a novel mechanism for constitutive PI3K/Akt activation in gliomas.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathophysiology, Guangzhou Medical University, Guangzhou 510182, China.

ABSTRACT
The PI3K/Akt signaling pathway is frequently activated in various human cancer types and plays essential roles in development and progression of cancers. Multiple regulators, such as phosphatase and tensin homolog (PTEN) and PH domain leucine rich repeat protein phosphatases (PHLPP), have also found to be involved in suppression of the PI3K/Akt signaling pathway. However, how suppressive effects mediated by these regulators are concomitantly disrupted in cancers, which display constitutively activated PI3K/Akt signaling, remains puzzling. In the present study, we reported that the expression of miR-93 was markedly upregulated in glioma cell lines and clinical glioma tissues. Statistical analysis revealed that miR-93 levels significantly correlated with clinicopathologic grade and overall survival in gliomas. Furthermore, we found that overexpressing miR-93 promoted, but inhibition of miR-93 reduced, glioma cell proliferation and cell-cycle progression. We demonstrated that miR-93 activated PI3K/Akt signaling through directly suppressing PTEN, PHLPP2 and FOXO3 expression via targeting their 3'UTRs. Therefore, our results suggest that miR-93 might play an important role in glioma progression and uncover a novel mechanism for constitutive PI3K/Akt activation in gliomas.

No MeSH data available.


Related in: MedlinePlus